Introduction to Aptamers

Since its advent in 1975, monoclonal antibody technology has become an important tool for basic scientific research and has been widely used in drug development, biosensors and other fields, with far-reaching medical significance. The world's first therapeutic antibody was discovered in 1986 and used to prevent kidney transplant rejection. Since then, many antibody drugs have been introduced and successfully used to treat a variety of diseases, such as asthma. However, the application of monoclonal antibodies in disease treatment still has certain limitations. In particular, for targeted therapy of lipids, carbohydrates and organic macromolecules, the affinity of antibodies is usually low, and the affinity when coupled with drugs is also easily affected. For this reason, artificial ligand-aptamer technology has gradually become a new research hotspot. As an effective supplement to traditional monoclonal antibodies, it has shown higher flexibility and broad application prospects.

Aptamers are a class of molecules composed of single-stranded nucleic acids that can specifically bind to a variety of targets. Compared with traditional antibodies, aptamers have unique advantages such as small size, low synthesis cost, uniform structure, and easy customization and modification. In addition, as nucleic acid templates, their stability and controllability also make them have significant potential in drug development. Aptamers have a wide range of applications and can cover small molecules, ions and other targets that are difficult for traditional antibodies to recognize. They have become a powerful artificial ligand supplement and are gradually emerging in multiple fields.

Nucleic acid aptamers are a type of single-stranded DNA or RNA molecules with highly specific molecular recognition capabilities, which are screened in vitro using SELEX technology (systematic evolution of ligand enrichment technology). These aptamers consist of 20 to 110 nucleotides, and their sequences include random regions and fixed regions. Through self-folding, aptamers can form small molecule groups with tertiary structures, have high affinity and can specifically bind to target molecules.

Advantages of nucleic acid aptamers: ① They have the advantages of high thermal stability, easy chemical synthesis and modification, and low immunogenicity, and are used in bioanalysis, biomedicine, biotechnology, sensor technology and other fields. ② They have the advantages of short production time, low cost and high specificity, and have broad application prospects in the field of orthopedics.

Disadvantages of nucleic acid aptamers: time-consuming and labor-intensive screening, high failure rate and high cost.

Method for in Vitro Screening of Aptamers: SELEX

SELEX is a method for in vitro screening of aptamers. Its principle is as follows:

(1) Design of Nucleic Acid Ligand Library

The nucleic acid ligand library can be prepared by chemical synthesis or biosynthesis.

(2) Affinity Screening

The nucleic acid ligand library is mixed and incubated with the target molecule, and the nucleic acid/target molecule complex is separated from the free nucleic acid sequence by solid phase adsorption, magnetic bead method, filtration method, gel electrophoresis and other methods.

(3) Elution

The nucleic acid/target molecule complex is obtained, and the nucleic acid sequence bound to the target molecule is released.

(4) PCR Amplification

PCR amplification is performed to obtain a nucleic acid library for the next round of screening.

(5) Cyclic Screening

Repeat the above steps for multiple rounds of screening to increase the proportion of nucleic acid sequences bound to the target molecule.

Fig.1 Schematic model of aptamer screening technology

Research Progress of SELEX Method

The field of nucleic acid aptamers has developed rapidly, and the target range of SELEX technology has expanded from the initial purified proteins and organic small molecules to almost all types of targets such as cells, viruses, tissue sections, ions, etc.

Negative-SELEX, Counter-SELEX, and Subtractive-SELEX are the conventional technical steps in the current screening technology to improve the specificity of nucleic acid aptamers. With the sharp decline in the price of second-generation sequencing, the second-generation sequencing technology of High-throughput-SELEX has become a conventional technical step in the current research of nucleic acid aptamer screening process and candidate aptamer screening. MB-SELEX and Cell-SELEX are widely used in the screening of nucleic acid aptamers specific to protein targets and tumor cells. Agarose gel microsphere-SELEX and MB-SELEX are often used for small molecule target nucleic acid aptamer screening.

In recent years, Capture-SELEX and GO-SELEX have been increasingly used, especially the optimized Capture-SELEX. SOMAmer-SELEX requires the use of chemically modified libraries; M-SELEX and Particle Display-SELEX require non-commercial microfluidic devices; CE-SELEX requires complex separation condition optimization and devices. These limitations make these methods less popular than the other methods mentioned above. In recent years, the development trend of M-SELEX technology is towards integration and miniaturization, but the screening efficiency needs to be improved. Since CE-SELEX was first reported in 2004, a series of improved methods have been reported.

According to the classification of methods for separating binding sequences from unbound sequences in the SELEX process, it can be divided into magnetic bead-based SELEX, library-immobilized SELEX, microfluidic-based SELEX, or capillary electrophoresis (CE)-based SELEX.

Challenges of SELEX Methods

After binding to cell receptors, aptamers are internalized and become targeted delivery agents for drugs. However, the therapeutic effect is affected by inherent physicochemical properties, such as metabolic instability, rapid renal filtration, rapid distribution from the plasma compartment to tissues (such as liver, spleen), nonspecific immune activation and polyanionic effects.

TekBiotech has successfully completed multiple nucleic acid aptamer projects, accumulated rich practical experience and mature technical systems, and can provide customers with efficient solutions. With in-depth research in the field of nucleic acid aptamers, the company has not only achieved remarkable results in project implementation, but also maintained a leading position in technology development and innovation. In addition, TekBiotech also provides a series of customized services, including the design and construction of nucleic acid aptamer libraries, expression and purification of antibodies, affinity determination, antibody sequence analysis and subsequent optimization. Through these services, the company can provide customers with full support from preliminary design to final product, ensuring that the personalized needs of different customers in scientific research and application are met.